This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Mitosis is an elaborate process where the duplicated genome is segregated into two daughter cells. Cytokinesis, the final step of mitosis, is the physical separation of the two daughter cells. Failure in cytokinesis can result in polyploidy and genome instability, two hallmarks found in many cancers. During cytokinesis, signaling by the anaphase spindle generates an equatorial zone of active RhoA, which in turn directs the cortical accumulation of contractile ring components. Rho family GTPases cycle between an active GTP-bound and inactive GDP-bound form. This cycle is controlled by GEFs, which promote GTP loading, and GAPs, which inactivate small GTPases by stimulating their low intrinsic GTPase activity. During cytokinesis, it has been proposed that localization of the RhoA GEF to the spindle midzone provides a means to locally activate RhoA at the cell equator. However, as RhoGTPases are anchored in the plasma membrane with a lipid moiety and are thought to freely diffuse within the membrane, previous theoretical work has suggested that a global RhoA GAP activity would be necessary to maintain a focused equatorial zone of active Rho. In C. elegans RGA-3 and RGA-4 (RGA-3/4), two highly similar RhoGAPs, were previously shown to act preferentially on RhoA and localizes to the cleavage furrow and the microtubule asters. We find that RGA-3/4 are required to restrict the accumulation of contractile ring components including anillin and the septins to the equator of the cell during early stages of cytokinesis. During cleavage furrow ingression RGA-3/4 are also important to limit the zone of anillin and the septins to a tight region at the furrow tip. Taken together our findings support a model in which a RhoGAP helps to restrict the activation of RhoA to the cell equator. RGA-3/4. To understand how RGA-3/4 activity and/or localization are regulated through mitosis we aim to identify new binding partners of RGA-3/4 using mass spectrometry after immunoprecipitation in collaboration with The Center for Physiological Proteomics.